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Citation: Death and Disease (2012) 3, e357; doi:10.1038/cddis.2012.95 & 2012 Macmillan Publishers Limited All rights reserved 2041-4889/12 www.nature.com/cddis AMP-activated -a1 as an activating kinase of TGF-b-activated kinase 1 has a key role in inflammatory signals

SY Kim1, S Jeong1, E Jung1, K-H Baik1, MH Chang1, SA Kim1, J-H Shim2, E Chun*,2 and K-Y Lee*,1

Although previous studies have proposed plausible mechanisms of the activation of transforming -b-activated kinase 1 (TAK1) in inflammatory signals, including Toll-like receptors (TLRs), its activating kinase still remains to be unclear. In the present study, we have provided evidences that AMP-activated (AMPK)-a1 has a pivotal role for activating TAK1, and thereby regulate NF-jB-dependent gene expressions in inflammatory signaling mediated by TLR4 and TNF-a stimulation. AMPK-a1 specifically interacts with TAK1 and reciprocally regulates their kinase activities. Upon the stimulation of lipopolysaccharide, AMPK-a1-knockdown (AMPK-a1KD) or TAK1-knockdown human monocytic THP-1 cells exhibit a dramatic reduction in the TAK1 or AMPK-a1 kinase activity, respectively, and subsequent suppressions of its downstream signaling cascades, which further leads to inhibitions of NF-jB and thereby productions of proinflammatory , such as TNF-a, IL-1b, and IL-6. Importantly, the microarray analysis of AMPK-a1KD cells revealed a dramatic reduction in the NF-jB-dependent genes induced by TLR4 and TNF-a stimulation, and the observation was in significant correlation with the results of quantitative real-time PCR. Moreover, AMPK-a1KD cells are highly sensitive to the TNF-a-induced , which is accompanied with dramatic reductions in the NF-jB-dependent and anti-apoptotic genes. As a result, our data demonstrate that AMPK-a1asan activating kinase of TAK1 has a key role in mediating inflammatory signals triggered by TLR4 and TNF-a. Cell Death and Disease (2012) 3, e357; doi:10.1038/cddis.2012.95; published online 26 July 2012 Subject Category:

Transforming growth factor-b (TGF-b)-activated kinase 1 catalytic (a) subunit and two non-catalytic regulatory subunits, (TAK1) is a member of the -activated (30 kDa) and g (38–63 kDa). There are several isoforms for kinase kinase family and a /threonine protein kinase.1–4 each of the three AMPK subunits, including a1, a2, b1, b2, g1, TAK1 functions as an upstream signaling molecule of NF-kB g2, and g3.11,12 The two AMPKa variants, a1 and a2, have and MAPKs in proinflammatory signals transduced by TNF-a, been shown to have a differential localization pattern in IL-1b, and Toll-like (TLR) ligands.5,6 Binding mammalian cells, with the AMPK-a1 subunit being localized in of lipopolysaccharide (LPS) to TLR4 causes recruitment of the , whereas the AMPK-a2 subunit localized in the MyD88, IRAK, and TRAF6 to the receptor.7,8 The TRAF6 nucleus.13,14 In terms of cell type specificity, most of the cells catalyzes synthesis of K63-linked polyubiquitin chains that express both AMPK-a1 and AMPK-a2 isoforms, whereas serve as a scaffold to recruit the TAK1 and IkB kinase (IKK) lymphcytes only express the AMPK-a1 isoform.15,16 A complexes. Recruitment of the kinase complexes facilitates previous report has addressed the relationship between T-cell autophosphorylation of TAK1 and subsequent phosphoryla- receptor-mediated signaling and AMPK-a1 through Ca2 þ - tion of IKKb by TAK1, leading to IkB degradation and -dependent protein kinase (CaMKK)-dependent subsequent activation of NF-kB.5,6 Despite of the plausible pathway, implying that AMPK-a1 may have specific roles in mechanisms by which TAK1 is activated,6 the existence of a immune cells.15 specific upstream kinase of TAK1 in the signaling cascades Here, we demonstrate that AMPK-a1 has a pivotal role in is still ambiguous. inducing proinflammatory signals, such as TLR4 and TNF-a, The AMP-activated protein kinase (AMPK) is a serine/ through the activation of TAK1. The knockdown of AMPK-a1 threonine protein kinase that has emerged as a master results in the marked reduction of TAK1 activity, and thereby of cellular energy balance in mammalian cells.9,10 The AMPK suppressions of downstream signaling cascades and the protein exists as a heterotrimer composed of a 63-kDa expression of NF-kB-dependent genes in response to LPS

1Department of Molecular Cell and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea and 2Department of and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, Boston, MA, USA *Corresponding authors: E Chun, Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, 665 Huntington Avenue, Building 1, Room 904, Boston, MA 02115, USA. Tel: 617 432 3250; Fax: 617 432 3259; E-mail: [email protected] or K-Y Lee, Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chenocheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea. Tel: +82 31 299 6225; Fax: +82 31 299 6229; E-mail: [email protected] Keywords: AMP-activated protein kinase-a1; TGF-b-activated kinase 1; NF-kB; Toll-like receptor 4; TNF-a-induced apoptosis Abbreviations: TAK1, transforming growth factor-b (TGF-b)-activated kinase 1; TLR, Toll-like receptor; IKK, IkB kinase; AMPK, AMP-activated protein kinase; LPS, lipopolysaccharide; AP-1, activator protein 1 Received 30.4.12; revised 12.6.12; accepted 20.6.12; Edited by T Brunner AMPK-a1 as an activating kinase of TAK1 SY Kim et al 2

stimulation, indicating an AMPK-a1-TAK1-NF-kB axis in the activation of AMPK.17–20 We raised a possibility of the TLR4-mediated signaling. Moreover, AMPK-a1-knockdown functional regulation between TAK1 and AMPK-a1 serine/ cells are highly sensitive to TNF-a-induced apoptosis, threonine protein kinase. We first tested the molecular supporting the functional role of AMPK-a1 associated interaction between AMPK-a1 and TAK1. The overexpressed with expressions of anti-apoptotic genes regulated by the TAK1 was significantly co-immunoprecipitated with over- TAK1-induced NF-kB activity. Thus, our results suggest that expressed AMPK-a1, and the interaction was enhanced by AMPK-a1 may be one of the upstream capable LPS stimulation (Figure 2a, lane 4 versus lane 5). To identify of inducing the activation of TAK1 in proinflammatory signals. the interaction domain in each other, TAK1 or AMPK-a1 truncated mutants were constructed (Supplementary Results Figure 2), and then immunoprecipitation assay was per- formed. Overexpressed AMPK-a1 was specifically co-immu- Inhibition of AMPK-a1 activity attenuates TLR4-mediated noprecipitated with all the truncated TAK1 construct, signaling pathway. Previous reports have demonstrated indicating that AMPK-a1 interacts with TAK1 through the the functional relationship between AMPK and TAK1;17–20 N-terminus of TAK1 (Figure 2b). In addition, TAK1 specifi- however, the functional role of AMPK-a1 has not yet been cally interacted with the autoinhibitory domain of AMPK-a1, elucidated in innate signal. Therefore, we have explored this AMPK-a1 (312–392) (Figure 2c). To verify the in vitro issue in human monocytic THP-1 cells with a TLR4 agonist, interaction, we further performed endogenous immunopreci- LPS. To know whether LPS stimulation induces the activa- pication assay. Consistent with in vitro interaction, endogen- tion of AMPK-a1, because it has been known that AMPK-a1, ous TAK1 was specifically coprecipitated with endogenous but not AMPK-a2, is solely expressed in human monocytic AMPK-a1, and the interaction was significantly enhanced in cells,16 THP-1 cells were treated with LPS for different time response to LPS stimulation (Figure 2d, lane 1 versus intervals and then the activation of AMPK-a1 was evaluated lane 2). These results suggest that the autoinhibitory domain with anti-pho-AMPK . The of AMPK-a1 interacts with the N-terminus of TAK1 of AMPK-a1 was gradually increased by LPS treatment in a (Figure 2e). We next explored whether AMPK-a1 is directly time-dependent manner (Supplementary Figure 1). To able to activate TAK1 in response to LPS stimulation. further examine whether AMPK-a1 is either a positive or To find an answer for our query, we generated knockdown negative regulator in the TLR4-mediated signaling pathway, THP-1 cells against AMPK-a1 and TAK1 protein by using leading to activate NF-kB and activator protein 1 (AP-1), siRNA-contained lentiviruses (Supplementary Figure 3 and THP-1 cells were stimulated with LPS in the presence or Figure 2f). Interestingly, of Thr172/174 absence of different concentrations of compound C, which is and Ser 485 of APK-a1 were markedly reduced and a pharmacological inhibitor of AMPK.21 Both NF-kB and the phosphorylation of TAK1 was dramatically reduced in KD AP-1 reporter activities induced by LPS were significantly AMPK-a1-knockdown (AMPK-a1 ) THP-1 cells treated with reduced in compound C-treated THP-1 in a dose-dependent LPS as compared with that of wt THP-1 cells (Figure 2f, KD KD manner (Figure 1a, NF-kB; Figure 1b, AP-1). Furthermore, AMPK-a1 ). Interestingly, TAK1-knockdown (TAK1 ) cells the inhibition of AMPK-a1 activity resulted in a dramatic exhibited marked reduction in phosphorylation of Thr172/174 reduction in the levels of proinflammatory cytokines, such as and Ser 485 of AMPK-a1 in response to LPS stimulation KD TNF-a, IL-6, and IL-1b, induced by LPS (Figure 1c, IL-6; (Figure 2f, TAK1 ). It was partly consistent with the Figure 1d, TNF-a; Figure 1e, IL-1b). We next attempted to previous report that TAK1 is involved in the activation of 17 verify the specific function of AMPK-a1 in terms of its kinase AMPK-a1. Consistent with the result, the kinase activity activity. For this, we constructed dominant negative (DN), of TAK1 or AMPK-a1 induced by LPS stimulation was KD KD AMPK-a1 (D159A), and constitutive active (CA), AMPK-a1 also significantly reduced in AMPK-a1 or TAK1 THP-1 (T174D), forms of AMPK-a1, and examined their functional cells, respectively, as compared with that of wt THP-1 cells roles (Figure 1f). Overexpression of wild-type (wt) AMPK-a1 (Figure 2g, TAK1; Figure 2h, AMPK-a1). These results slightly enhanced the activities of both, NF-kB and AP-1, in strongly demonstrate that the autoinhibitory domain of the presence or absence of LPS stimulations (Figures 1g AMPK-a1 interacts with N-terminus of TAK1, which further and h, closed bar), whereas AMPK-a1 (D159A) DN reciprocally regulates their kinase activities (Figure 2e). significantly suppressed these activities (Figures 1g and h, chute-block bar). In contrast, AMPK-a1 (T174D) CA induced AMPK-a1-knockdown THP-1 cells exhibit the impair- increase in the activities of NF-kB and AP-1 (Figures 1g and ments of TLR4-mediated signaling pathway. To under- h, hatched bar). Consistent with the above-mentioned stand the functional role played by AMPK-a1 in TLR4- findings, similar results in the production of inflammatory mediated signaling pathway, biochemical studies were KD cytokines were observed (Figure 1i, TNF-a; Figure 1j, IL-6; performed in AMPK-a1 THP-1 cells. Basal activities of KD Figure 1k, IL-1b). These results suggest that AMPK-a1 may NF-kB and AP-1 were significantly decreased in AMPK-a1 be positively involved in TLR4-mediated signaling pathway. THP-1 cells, as compared with that of wt THP-1 cells (Figures 3a and b, closed bars). Their activity were markedly The autoinhibitory domain of AMPK-a1 interacts with increased in LPS-treated wt THP-1 cells (Figures 3a and b, N-terminus of TAK1, and that reciprocally regulate their doted bars), moreover, significantly enhanced in wt-AMPK- activations. We next explored the molecular mechanism by a1-expressed wt THP-1 stimulated with LPS, as compared which AMPK-a1 is involved in TLR4-mediated signaling. with that of unstimulated wt THP-1 cells (Figures 3a and b, Previous reports have shown that TAK1 is involved in chute-block bar). To gain further direct evidence on whether

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 3

Figure 1 Inhibition of AMPK-a1 activity attenuates TLR4-signaling pathway. (a and b) The THP-1 cells were transfected with either pBIIx-Luc (a) or AP-1-luc (b) together with Renilla luciferase vector. At 24 h after transfection, cells were treated with or without LPS (10 ng/ml) for 6 h in the presence or absence of different concentrations of compound C as indicated, and then analyzed for luciferase activity. Results are expressed as the fold induction in luciferase activity relative to that of untreated cells. Error bars indicate ±S.D. of triplicate samples. (c–e) The THP-1 cells were treated with or without LPS (10 ng/ml) for 9 h in the presence or absence of different concentrations of compound C as indicated, and then analyzed for productions of IL-6 (c), TNF-a (d), and IL-1b (e) in supernatants using ELISA method. Error bars indicate ±S.D. of triplicate samples. (f) The AMPK-a1 (D159A) and AMPK-a1 (T174D) mutants were generated by site-directed mutagenesis as described in Materials and Methods. (g and h) The THP-1 cells were transfected with mock, wt (wt) hAMPK-a1, DN hAMPK-a1 D159A, or constitutive active (CA) hAMPK-a1 T174D vector. At 24 h after transfection, cells were transfected with either pBIIx-Luc (g) or AP-1-luc (h) together with Renilla luciferase vector. After 24 h, cells were treated with or without LPS (10 ng/ml), and then analyzed for luciferase activity. Results are expressed as the fold induction in luciferase activity relative to that of untreated cells. Error bars indicate ±S.D. of triplicate samples. (i–k) The THP-1 cells were transfected with mock, wt hAMPK-a1, DN hAMPK-a1 D159A, or CA hAMPK-a1 T174D vector. At 24 h after transfection, cells were treated with or without LPS (10 ng/ml) for 9 h, and then analyzed for productions of TNF-a (i), IL-6 (j), and IL-1b (k) in supernatants using ELISA method. Error bars indicate ±S.D. of triplicate samples. *Po0.05, **Po0.01, NS, not significant

AMPK-a1 is involved in the TLR4-mediated signal, we with AMPK-a1 vector were treated with or without LPS. performed the rescue experiment in AMPK-a1KD THP-1 Interestingly, the activities of NF-kB and AP-1 were markedly cells. The wt AMPK-a1 vector was transfected in the AMPK- recovered, as compared with that of mock transfectant a1KD THP-1 cells. The AMPK-a1KD THP-1 cells transfected AMPK-a1KD THP-1 cells treated with LPS (Figures 3a and b

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 4

Figure 2 Molecular interaction between AMPK-a1 and TAK1. (a) The HEK293 cells were transfected with -TAK1, Flag-AMPK-a1, or Myc-TAK1 and Flag-AMPK-a1 vector. At 36 h after transfection, cells were treated with or without LPS (100 ng/ml) for 45 min. The cells were extracted and immunoprecipitated with anti-Myc antibody. The interaction was detected by western blotting with anti-Flag antibody. The presence of Myc-TAK1 and Flag-AMPK-a1 in the pre-IP lysates was verified by western blot analysis. (b) The HEK293 cells were transfected with Flag-AMPK-a1 and wt Myc-TAK1, Flag-AMPK-a1 and Myc-TAK1 (1–500), Flag-AMPK-a1 and Myc-TAK1 (1–400), Flag-AMPK-a1 and Myc-TAK1 (1–300), Flag-AMPK-a1 and Myc-TAK1 (1–200), or Flag-AMPK-a1 and Myc-TAK1 (1–100) vectors. At 36 h after transfection, transfected cells were extracted and immunoprecipitated with anti-Flag antibody. The interaction was detected by western blotting with anti-Myc antibody. The presence of Myc-TAK1 mutants and Flag-AMPK-a1 in the pre-IP lysates was verified by western blot analysis. (c) The HEK293 cells were transfected with Myc-TAK1 and wt Flag-AMPK-a1, Myc-TAK1 and Flag-AMPK-a1 (1–312), or Myc-TAK1 and Flag-AMPK-a1 (1–392). At 36 h after transfection, transfected cells were extracted and immunoprecipitated with anti-Myc antibody. The interaction was detected by western blotting with anti-Flag antibody. The presence of Flag-AMPK-a1 mutants and Myc-TAK1 in the pre-IP lysates was verified by western blot analysis. (d) The THP-1 cells were treated with or without LPS (100 ng/ml) for 45 min. Cells were extracted and immunoprecipitated with anti-TAK1 antibody. The interaction was detected by western blotting with anti-AMPK-a1 antibody. The presence of TAK1 in the pre-IP lysates was verified by western blot analysis. (e) A model of molecular interaction between AMPK-a1 and TAK1. Above results suggest the molecular interaction between the autoinhibitory domain of AMPK-a1 and the N-terminus of TAK1. (f) WT THP-1, AMPK-a1KD, and TAK-1KD THP-1 cells were treated with or without LPS (10 ng/ml) for different times as indicated. Cell lysates were subjected to western blot analysis using the indicated . (g and h) WT THP-1, AMPK-a1KD, and TAK-1KD THP-1 cells were treated with or without LPS (10 ng/ml) for different times as indicated. In vitro kinase assay for TAK1 (g) and AMPK-a1(h) was performed as described in Materials and Methods. Error bars indicate ±S.D. of triplicate samples. *Po0.05, **Po0.01

in AMPK-a1KD, dotted bar versus chute-block bar). Given THP-1 cells (Figure 3g, pho-TAK1). Interestingly, phosphor- these results, we further assessed DNA-binding activities of ylations of other downstream signaling molecules, such as NF-kB, p65 and p50, and AP-1, c-fos and c-jun. Consistently, p-38, JNK, and AKT, were also significantly decreased in overexpressions of AMPK-a1 in wt THP-1 markedly AMPK-a1KD THP-1 cells (Figure 3g). These results were enhanced DNA-binding activities of p65, p50, c-fos, and partly consistent with previous reports that AMPK has an c-jun (Figure 3c, p65; Figure 3d, p50; Figure 3e, c-fos; important role in promoting activations of p38 MAPK, JNK, Figure 3f, c-jun). Furthermore, these DNA-binding activities and AKT.22–24 Moreover, we found that a marked reduction were significantly increased in AMPK-a1-transfected AMPK- of proinflammatory cytokines, such as TNF-a, IL-1b, and a1KD THP-1 cells (Figure 3c, p65; Figure 3d, p50; Figure 3e, IL-6, induced by LPS stimulation could be detected in c-fos; Figure 3f, c-jun), indicating a positive role of AMPK-a1 AMPK-a1KD THP-1 cells, as compared with that of wt in TLR4-mediated signaling. Based on the functional role of THP-1 cells (Figure 3h). These results provide evidence on AMPK-a1 in TLR4-mediated activations of NF-kB and AP-1, the positive role of AMPK-a1 in TLR4-mediated signaling we next assessed regulations of signaling molecules, which pathway through the activation of TAK1. appear in the TLR4-mediated signaling cascades. For this, wt THP-1 and AMPK-a1KD THP-1 cells were treated with AMPK-a1-knockdown THP-1 cells exhibit the impair- or without LPS for different times. As expected, the ments of expression of NF-kB-dependent genes induced phosphorylation of TAK1 was markedly attenuated in by TLR4. To validate the axis of AMPK-a1-TAK1-NF-kBin AMPK-a1KD THP-1 cells, as compared with that of wt TLR4-mediated signaling pathway in a detailed manner, we

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 5

Figure 3 AMPK-a1-knockdown THP-1 cells exhibit impairments of TLR4-mediated signal, leading to activation of NF-kB and AP-1. (a and b) The wt THP-1 and AMPK- a1KD THP-1 cells were transfected with or without hAMPK-a1 vector. Twenty-four hours after transfection, cells were transfected with either pBIIx-Luc (a) or AP-1-luc (b) together with Renilla luciferase vector. After 24 h, cells were treated with or without LPS (10 ng/ml) for 6 h, and then analyzed for luciferase activity. Results are expressed as the fold induction in luciferase activity relative to that of untreated cells. Error bars indicate ±S.D. of triplicate samples. (c–f) The wt THP-1 and AMPK-a1KD THP-1 cells were transfected with or without hAMPK-a1 vector. Twenty-four hours after transfection, cells were treated with or without LPS (10 ng/ml) for 1 h, and then analyzed for DNA-binding activities for NF-KB, p65 (c) and p50 (d), and AP-1, c-Fos (e) and c-Jun (f), components as described in Materials and Methods. Results are expressed as the fold increase relative to that of wt THP-1 transfected with mock. Error bars indicate ±S.D. of triplicate samples. (g) The wt THP-1 and AMPK-a1-knockdown (AMPK-a1KD) THP-1 cells were treated with or without LPS (10 ng/ml) for different times as indicated. The lysates were examined by western blotting with anti-pho-TAK1, anti-TAK1, anti-IkBa, anti-pho- p38, anti-p38, anti-pho-JNK, anti-JNK, anti-pho-AKT, and anti-AKT antibodies. Immunoblotting with anti-GAPDH antibody was performed to generate a control for gel loading. (h) The wt THP-1 and AMPK-a1-knockdown (AMPK-a1KD) THP-1 cells were treated with or without LPS (10 ng/ml) for 9 h, and then analyzed for productions of TNF-a, and IL-1b, and IL-6 in supernatants using ELISA method. Error bars indicate ±S.D. of triplicate samples. *Po0.05, **Po0.01, ***Po0.001

performed analysis. According to stimula- group 2 genes were greatly upregulated in LPS-treated wt tion of LPS, marked changes in gene expression profiles THP-1 cells, as compared with that of without stimulation, could be detected (Supplementary Figure 4). To assess the whereas minute changes could be detected in LPS-treated NF-kB-dependent gene expressions by LPS stimulation, 94 AMPK-a1KD THP-1 cells, as compared with that of without genes containing specific kB-binding DNA sequences were stimulation (Figure 4c, group 1 gene; Figure 4d, group further sorted out. Expression levels of 94 genes were 2 gene). These results strongly support that AMPK-a1is significantly altered in either wt or AMPK-a1KD THP-1 cells by positively involved in the TLR4-mediated signaling pathway treatment with or without LPS (Figure 4a). According to through the activation of TAK1, and thereby regulates the relative up and down expressions in each combination, TLR4-mediated NF-kB-dependent gene expression. 36 genes were selected, and were subdivided into two groups: group 1, highly regulated genes such as IL-1b, IL-8, AMPK-a1 is functionally involved in TNF-a-induced CD44, TNF-a, and CCL-5; and group 2, moderately regulated apoptosis through the regulation of NF-kB-dependent genes such as NF-KB1, NF-KB1A, NF-KB1E, RELB, genes. Previous studies have shown that TAK1 has a IER3, REL, and BCL3 (Figure 4b, Supplementary Table 1a, protective role against TNF-a-induced apoptosis through the and Supplementary Table 1b). To verify their expression, we activation of NF-kB.3,4 In addition, a recent report has shown performed quantitative real-time PCR (qRT-PCR) analysis that AMPK confers protection against TNF-a-induced cardiac with specific primers targeted to each gene. Both group 1 and cell death.25 Based on these previous reports and our above

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 6

Figure 4 AMPK-a1-knockdown THP-1 cells exhibit a marked reduction in NF-kB-dependent genes in response to LPS stimulation. (A) The wt THP-1 and AMPK-a1KD THP-1 cells were treated with or without LPS (100 ng/ml) for 3 h. Total RNAs were isolated from each sample and microarray analysis was performed as described in Materials and Methods. The96NF-kB-dependent upregulated and downregulated genes were sorted and represented: a, wt THP-1 cells treated with LPS versus wt THP-1 cells; b, AMPK-a1KD THP-1 cells treatedwithLPSversus AMPK-a1KD THP-1 cells; c, AMPK-a1KD THP-1 cells versus wt THP-1 cells; and d, AMPK-a1KD THP-1 cells treated with LPS versus wt THP-1 cells treated with LPS. (B) Among 96 NF-kB-dependent genes, 36 genes were further selected and their expression patterns were represented. *, moderately regulated genes; **, highly regulated genes. (C) The WT THP-1 and AMPK-a1KD THP-1 cells were treated with or without LPS (100 ng/ml) for 3 h. Total RNAs were isolated from each sample and quantitative RT-PCR analysis was performed with specific primers targeted to IL-8, TNF, IL-1b, CD44, and CCL5 genes. Error bars represent mean±S.D. of triplicate samples. (D) The wt THP-1 and AMPK-a1KD THP-1 cells were treated with or without LPS (100 ng/ml) for 3 h. Total RNAs were isolated from each sample and quantitative RT-PCR analysis was performed with specific primers targeted to NF-KB1, NF-KB1A, NF-KB1E, RELB, IER3, REL, and BCL3 genes. Error bars represent mean±S.D. of triplicate samples

Figure 5 AMPK-a1-knockdown THP-1 cells are highly sensitive to TNF-a-induced apoptosis. (a and b) TAK1 À / À MEF cells were transfected with mock or Myc-TAK1 vector. At 24 h after trasnfection, cells were treated without (a) or with (b) TNF-a (100 ng/ml) for 24 h, and then stained with BD Cycletest Plus-DNA reagent in accordance with the manufacturer’s protocol. The stained cells were analyzed with FACSCalibur system and apoptotic cell death was determined with Modfit LT 3.0 software. Error bars represent mean±S.D. of three independent experiments. (c and d) The wt THP-1 (c) and AMPK-a1KD THP-1 cells (d) were cultured with culture medium containing 2% FBS for 24 h, and then treated with or without TNF-a (100 ng/ml) for different times, as indicated. Cells were harvested and stained with FITC V Apoptosis Detection Kit (BD Biosciences) in accordance with the manufacturer’s protocol. Samples were analyzed with FACSCalibur system and apoptotic cells were determined with CellQuest software. Error bars represent mean±S.D. of three independent experiments. (e–g) The wt THP-1 and AMPK-a1KD THP-1 cells were cultured with culture medium containing 2% FBS for 24 h, and then treated with or without TNF-a (100 ng/ml) for different times, as indicated. -3 (e), caspase-8 (f), and caspase-9 (g) activities were measured using the CaspACE according to the manufacture’s instructions. Error bars represent mean±S.D. of three independent experiments. *Po0.01. (h) The genomic DNA extracts were prepared as described in Materials and Methods, run on 1.8% agarose gels, and visualized under UV illumination

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 7 results, we assumed that, if AMPK-a1 has a key role in the expected, apoptotic cell death induced by TNF-a was activation of TAK1, and thereby regulates the expression of markedly increased in TAK1 À / À MEF cells (Figures 5a NF-kB-dependent genes, the defectiveness of AMPK-a1 and b, 9±2% in mock without TNF-a versus 23±4% in might be critically affected on TNF-a-induced apoptosis. As mock with TNF-a), whereas the apoptosis was significantly

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 8

decreased in TAK1-overexpressed TAK1 À / À MEF cells than in the case of mock transfectants (Figure 5b, 23±4% in mock with TNF-a versus 10±3% in TAK1 with TNF-a). Interestingly, AMPK-a1KD THP-1 cells were highly sensitive to TNF-a-induced apoptosis in a time-dependent manner as compared with that of wt THP-1 cells (Figure 5c, wt THP-1; Figure 5d, AMPK-a1KD). To verify the results, we performed caspase activity and DNA laddering assay in the same condition. Consistently, the activities of caspase 3, caspase 9, and were markedly higher in AMPK-a1KD THP- 1 cells than that in wt THP-1 cells activities (Figure 5e, caspase 3; Figure 5f, caspase 9; Figure 5g, caspase 8). Moreover, similar results could be seen in DNA laddering assay (Figure 5h). These results suggest that AMPK-a1is functionally involved in TNF-a-induced apoptosis through the regulation of TAK1 activity. TNF-a signaling is transduced through its receptors to simultaneously elicit two opposing effects: the induction of apoptosis and the of anti-apoptotic genes.26,27 We therefore examined whether the effects of AMPK-a1on TNF-a-induced apoptosis are linked to the expression of NF-kB-dependent genes and pro-apoptotic or anti-apoptotic genes; we performed gene expression analysis of wt and AMPK-a1KD THP-1 cells with or without TNF-a stimulation, respectively. According to stimulation of TNF-a, marked changes in gene expression profiles could be detected (Supplementary Figure 5). When we compared gene-expres- sion profiles related to NF-kB-dependent genes, marked changes could also be observed (Figure 6a, Supplementary Table 2a and Supplementary Table 2b). qRT-PCR analysis demonstrated that five different genes such as IL-1b, IL-8, TNF-a, CCL5, and CD44 were greatly upregulated in TNF-a- treated wt THP-1 cells as compared with that of without stimulation, whereas minute changes could be detected in TNF-a-treated AMPK-a1KD THP-1 cells, as compared with that of without stimulation (Figure 6b). Similar results could also be detected in qRT-PCR analysis of NF-KB1A, NF- KB1A, NF-B1E, REL, BCL3, and IER3 (Supplementary Figure 6), indicating that AMPK-a1 is functionally related to the expression of NF-kB-dependent genes in response to TNF-a stimulation.

Suppression of AMPK-a1 is associated with the expres- sion of pro- or anti-apoptotic genes in response to TNF-a stimulation. We further assessed whether the sensitivity on TNF-a-induced apoptosis in AMPK-a1KD cells is associated with the gene expression profiles related to apoptosis. Figure 6 AMPK-a1-knockdown THP-1 cells exhibit a marked reduction in According to stimulation of TNF-a, marked changes in both NF-kB-dependent genes in response to TNF-a stimulation. (A) The wt THP-1 and KD pro-apoptotic and anti-apoptotic gene expression profiles AMPK-a1 THP-1 cells were treated with or without TNF-a (100 ng/ml) for 12 h. could be detected (Figure 7a, Supplementary Tables 3a, 3b Total RNAs were isolated from each sample and microarray analysis was performed as described in Materials and Methods. The 96 NF-kB-dependent upregulated and and 4a, 4b). To verify these gene expression profiles, we downregulated genes were sorted and represented: a, wt THP-1 cells treated with selected four different pro-apoptotic genes, such as PTEN, TNF-a versus wt THP-1 cells; b, AMPK-a1KD THP-1 cells treated with TNF-a versus LTA, MAGED1, and TNFRFS21, and four different anti- AMPK-a1KD THP-1 cells; c, AMPK-a1KD THP-1 cells versus wt THP-1 cells; and d, apoptotic genes, such as SGK1, PI3KCB, BCL2L2, and AMPK-a1KD THP-1 cells treated with TNF-a versus wt THP-1 cells treated with KD BCL2, and performed qRT-PCR analysis. The pro-apoptotic TNF-a.(B) The wt THP-1 and AMPK-a1 THP-1 cells were treated with or without genes were markedly increased in AMPK-a1KD cells non- TNF-a (100 ng/ml) for 12 h. Total RNAs were isolated from each sample and quantitative RT-PCR analysis was performed with specific primers targeted to IL-8, treated with TNF-a, as compared with that of wt THP-1 cells ± KD TNF-a, IL-1b, CD44, and CCL5 genes. Error bars represent mean S.D. (Figure 7b, AMPK-a1 versus wt THP-1 in the group of triplicate samples without stimulation of TNF-a). According to stimulation of TNF-a, moreover, two of them, PTEN and LTA, were

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 9

Figure 7 TNF-a-induced apoptosis in AMPK-a1-knockdown THP-1 cells is associated with the expression of pro- or anti-apoptotic genes. (A) The wt THP-1 and AMPK- a1KD THP-1 cells were treated with or without TNF-a (100 ng/ml) for 12 h. Total RNAs were isolated from each sample and microarray analysis was performed as described in Materials and Methods. The pattern of pro-apoptotic and anti-apoptotic gene expression was represented; a, wt THP-1 treated with TNF-a versus wt THP-1; b, AMPK-a1KD THP-1 cells treated with TNF-a versus AMPK-a1KD THP-1 cells; c, AMPK-a1KD THP-1 cells versus wt THP-1; and d, AMPK-a1KD THP-1 cells treated with TNF-a versus wt THP-1 cells. (B) The wt THP-1 and AMPK-a1KD THP-1 cells were treated with or without TNF-a (100 ng/ml) for 12 h. Total RNAs were isolated from each sample and quantitative RT-PCR analysis was performed with specific primers targeted to pro-apoptotic genes such as PTEN, LTA, MAGED1, and TNFRFS21 genes. Error bars represent mean±S.D. of triplicate samples. (C) The wt THP-1 and AMPK-a1KD THP-1 cells were treated with or without TNF-a (100 ng/ml) for 12 h. Total RNAs were isolated from each sample and quantitative RT-PCR analysis was performed with specific primers targeted to anti-apoptotic genes such as SGK1, PI3KCB, BCL2L2, and BCL2 genes. Error bars represent mean±S.D. of triplicate samples significantly enhanced in AMPK-a1KD cells (Figure 7b, upper NF-kB transcriptional activity.28,29 In contrast, the anti- panels). The obtained result was critically consistent with apoptotic genes, such as SGK1, PI3KCB, and BCL2L2, previous reports, which state that PTEN expression aug- were significantly enhanced by the stimulation of TNF-a in wt ments TNF-a-induced apoptosis through the inhibition of THP-1 cells, whereas no significant changes could be

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 10

detected in AMPK-a1KD cells (Figure 7c). Overall, these knockdown or wt THP-1 cells after TNF-a treatment, results strongly provide evidence that the TNF-a-induced furthermore, marked inductions of pro-apoptotic genes could apoptosis in AMPK-a1KD cells is associated with expressions be detected, whereas significant reductions in anti-apoptotic of pro- or anti-apoptotic genes related to NF-kB transcrip- genes could be detected in AMPK-a1-knockdown THP-1 tional activity. cells. These results were similar to that of TNF-a-induced apoptosis in TAK1 À / À MEF cells. Therefore, the obtained results support a pivotal role of AMPK-a1 as an upstream Discussion kinase of TAK1, which eventually lead to express NF-kB- Our experiments demonstrate that AMPK-a1 critically dependent genes related in the protection to TNF-a-induced regulates TLR4-mediated signaling pathway through the apoptosis. activation of TAK1. We found that autoinhibitory domain of Although it is not completely understood as how TAK1 AMPK-a1 interacts with the N-terminus of TAK1. The masking activity is biochemically regulated, its activation seems to of autoinhibitory domain of AMPK-a1 seems to be critically require TAK1-binding such as TAB1, TAB2, and associated with the activation of AMPK-a1 by TAK1,30 which TAB3. In addition, a plausible model about how to activate led us to speculate that AMPK-a1 might regulate the activity of TAK1 in innate signaling has been proposed in a recent TAK1 through the intermolecular interaction. In terms of the report.6 Binding of IL-1b to IL-1R or LPS to TLR4 causes activity of AMPK-a1, the inhibition of AMPK-a1 activity with a recruitment of adopter proteins such as MyD88, IRAK, and pharmacological compound, compound C, revealed a dra- TRAF6 to the receptor. The TRAF6 in turn catalyzes synthesis matic reduction in NF-kB and AP-1 activities, and production of K63-linked polyubiquitin chains. The polyubiquitin chains of proinflammatory cytokines, such as TNF-a, IL-6, and IL-1b, acts as a scaffold motif to recruit the TAK1 and IKK complexes in response to LPS stimulation. Moreover, we found that a through binding to the regulatory subunits TAB2 and NEMO, constitutive active form of AMPK-a1 significantly augmented respectively. Recruitment of the kinase complexes facilitates these activities induced by LPS treatment, but not in a DN autophosphorylation of TAK1 and subsequent phosphoryla- form as AMPK-a1. These results suggest that AMPK-a1is tion of IKKb by TAK1, thus leading to IkB degradation and functionally involved in TLR4-mediated signaling through the subsequent activation of NF-kB. Our data propose a potential molecular interaction with TAK1. possibility that TAK1 activity in both TLR4- and TNF-a- The TAK1 was originally identified as a TGF-b-activated mediated signaling pathway may be regulated by AMPK-a1as kinase.1–3 The TAK1 is involved in distinct cellular signals an upstream kinase of TAK1. Taken together, our data are such as Wnt, bone morphogenetic protein, activin, and TGF- summarized in Figure 8. Binding of LPS derived from bacterial b-signaling pathways.3,31,32 In addition, TAK1 has a major role components to TLR4 initiates intracellular-signaling cas- in both innate and adaptive immunity pathways.5–8 We found cades, initially leading to the recruitment of adaptor molecules that, in AMPK-a1-knockdown THP-1 cells, TAK1 phosphor- such as MyD88, IRAKs, and Mal proteins. Two critical events ylation was markedly reduced in response to LPS stimulation for the activation of TAK1 may occur in the middle of signaling when compared with that of wt THP-1 cells. In agreement with cascades. Upon the TLR4 stimulation, the activation of previous reports,22–24 phosphorylations of JNK, p38, and AKT AMPK-a1 is simultaneously induced by LKB1 and Ca2 þ - were impaired in LPS-treated AMPK-a1-knockdown THP-1 calmodulin-dependent protein kinase, and then activated cells, indicating that AMPK has an important role in promoting AMPK-a1 can induce activation of JNK/p38MAPK pathway, p38, JNK, AKT activations. Furthermore, DNA-binding activ- thereby inducing activations of AP-1 components such as ities of p65, p50, c-Fos, and c-Jun were markedly reduced in c-Jun and c-fos. Simultaneously, activated AMPK-a1 speci- AMPK-a1-knockdown THP-1 cells. In AMPK-a1-knockdown fically interacts with TAK1 through the molecular interaction THP-1 cells, dramatic reductions in TNF-a, IL-6, and IL-1b between N-terminus of TAK1 and autoinhibitory domain of could be detected in response to LPS stimulation. Moreover, AMPK-a1, and subsequently induces the phosphorylation of microarray and qRT-PCR analysis targeted to NF-kB-depen- TAK1 for the activation. In turn, the activated TAK1 subse- dent genes critically revealed marked reductions of the quently induces the phosphorylation of IKKb, leading to IkB expression of these genes in AMPK-a1-knockdown THP-1 degradation and activation of NF-kB. Nevertheless, the cells after LPS treatment. These results strongly support the existence of other upstream kinases capable of inducing hypothesis that AMPK-a1 is critically required for TLR4- TAK1 activation and the reciprocal regulation between TAK1 mediated signaling, thus leading to activation of NF-kB and and AMPK-a1 is not completely ruled out. Therefore, future AP-1, and thereby regulating expressions of NF-kB-depen- studies elucidating the specific molecular mechanism may dent genes. contribute towards more detailed understanding on the To verify the functional role of AMPK-a1 capable of functional roles played by TAK1 as a key element kinase in regulating TAK1 activity, we confirmed the effects in another different innate signaling pathways. system, TFN-a-induced apoptosis. Previous studies have shown that TAK1 has a protective role against TNF-a-induced apoptosis through the activation of NF-kB.3,4 Interestingly, Materials and Methods AMPK-a1-knockdown cells were highly sensitive to TNF-a- Cells and antibodies. The HEK 293 cells (human kidney embryonic cells) were purchased from American Type Culture Collection (ATCC, Manassas, induced apoptosis along with a marked reduction in the levels VA, USA) and maintained in DMEM medium (Invitrogen Corporation, Carlsbad, of NF-kB-dependent genes as compared with those of wt CA, USA) containing 10% FBS, 2 mM L-glutamine, 100 unit/ml of penicillin, 100 mg/ml THP-1 cells. When we analyzed the expression of anti- streptomycin, and 5 Â 10 À 5 M b-mercaptoethanol. The THP-1 cells (human apoptotic genes and pro-apoptotic genes in AMPK-a1- monocytic cells) were purchased from ATCC and maintained in RPMI medium

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 11

adding the appropriate empty vector to the DNA mixture. Typically 24 h after transfection, cells were lyzed and luciferase activity was measured using the dual luciferase assay kit (Promega).

Measurement of cytokines. Wt THP-1 cells were transiently transfected using Neon transfection system, according to the respective manufacturer’s instructions, with mock, WT hAMPK-al, hAMPK-a1 (D159A), and hAMPK-a1 (T174D) vectors. Cells were stimulated with or without LPS (10 ng/ml) for 9 h, and subsequently supernatants were harvested. Levels of TNF-a, IL-1b, and IL-6 were measured in the supernatants, according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). Wt THP-1 and AMPK-a1KD THP-1 cells were stimulated with or without LPS (10 ng/ml) for 9 h, and then supernatants were harvested. Levels of TNF-a, IL-1b, and IL-6 were measured in the supernatants, according to the manufacturer’s protocol (R&D Systems).

p65/p50 (NF-kB) and c-Fos/c-Jun (AP-1) DNA-binding assay by -linked immunosorbent assay. Nuclear proteins from transfec- tants treated with or without LPS were prepared with CelLyticTM NuCLEARTM Extraction kit in accordance with the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). Activities of transcription factors, p65, p50, c-Jun, and c-Fos, were determined with TransAM NF-kB or AP-1 assay kit according to the manufacturer’s instructions (Active Motif North America, Carlsbad, CA, USA).4,34 Briefly, a specific double-stranded DNA sequence containing the NF-kB or AP-1 response element was immobilized on a 96-well plate, and the binding of the activated p65 or p50 to the NF-kB response element and c-fos or c-Jun to the AP-1 response element was achieved by incubating nuclear extracts in the presence of binding buffer. The interaction between the protein–DNA complexes was detected by the addition of specific antibodies against p65, p50, c-fos, or pho-c-Jun. Addition of a secondary antibody conjugated to horseradish peroxidase provided a sensitive colorimetric readout at 450 nm. Figure 8 Schematic model showing role of AMPK-a1 as an upstream kinase of TAK1 in TLR4-mediated signaling pathway. Binding of LPS derived from bacterial Plasmids and mutagenesis. The pcDNA3-Flag-hAMPK-a1 wt was kindly components to TLR4 initiates intracellular signaling cascades, initially leading to the provided by Dr. SJUm.35 The pcDNA3-Myc-hTAK1 wt was generated by PCR, recruitment of adapter molecules such as MyD88, IRAKs, and Mal proteins. Two using HeLa cDNA library as a template, and inserted into pcDNA3. Myc-TAK1 critical events for the activation of TAK1 may occur in the middle of signaling mutants, Myc-TAK1 1–500, Myc-TAK1 1–400, Myc-TAK1 1–300, Myc-TAK1 1–200, cascades. Upon TLR4 stimulation, the activation of AMPK-a1 is simultaneously and Myc-TAK1 1–100, were generated by PCR, using Myc-TAK1 wt as a template, 2 þ induced by LKB1 and Ca -calmodulin-dependent protein kinase, and then and inserted into pcDNA3. The Flag-AMPK-a1 mutants, Flag-AMPK-a1 (1–312) activated AMPK-a1 can induce activation of JNK/p38MAPK pathway, thereby and Flag-AMPK-a1 (1–392), were generated by PCR, using Flag-AMPK-a1wtas inducing activations of AP-1 components such as c-Jun and c-fos. Simultaneously, a template, and inserted into pcDNA3. Flag-AMPK-a1 mutants, Flag-AMPK-a1 activated AMPK-a1 specifically interacts with TAK1 through the molecular (D159A) and Flag-AMPK-a1 (T174D) were generated using the MORPH plasmid interaction between N-terminus of TAK1 and autoinhibitory domain of AMPK-a1, DNA mutagenesis kit supplied by 50-30 Inc. (Boulder, CO, USA).36,37 and then AMPK-a1 and TAK1 reciprocally induces phosphorylations of TAK1 for the activation. In turn, the activated TAK1 subsequently induces the phosphorylation of Immunoprecipitation and western blot analysis. Transfected HEK IKKb, leading to IkB degradation and activation of NF-kB. Subsequently, 293 cells with appropriate expression vectors were lysed in a lysis buffer proinflammatory genes such as TNF-a, IL-1b, and IL-6 are expressed containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 1.5% aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Cellular

L debris was removed by centrifugation. Co-immunoprecipitation procedures were (Invitrogen Corporation) containing 10% FBS, 2 mM -glutamine, 100 unit/ml 4,36 of penicillin, 100 mg/ml streptomycin, and 5 Â 10 À 5 M b-mercaptoethanol. followed as previously described. For immunoprecipitation and western blotting, TAK1 À / À MEF cells were cultured in DMEM medium (Invitrogen Corporation) we used anti-Myc and anti-Flag antibodies (Sigma-Aldrich). The proteins were containing 5% FBS, 2 mM L-glutamine, 100 unit/ml of penicillin, and 100 mg/ml detected by the enhanced chemiluminescence system (Amersham Pharmacia streptomycin and 5 Â 10 À 5 M b-mercaptoethanol.3 Antibodies used were Biotech, Amersham, UK). For endogenous immunoprecipitation, THP-1 cells were anti-pho-AMPK-a1 (Ser485), anti-pho-AMPK-a1 (Thr172/Thr174), anti-pho-TAK1, treated with or without LPS (100 ng/ml) for 45 min. Cells were extracted and anti-TAK1, anti-IkBa, anti-pho-p38, anti-p38, anti-pho-JNK, anti-JNK, anti-pho-AKT, immunoprecipitated with anti-TAK1 antibody. The interaction was detected by anti-AKT, anti-Myc, anti-Flag, anti-GAPDH (, Beverly, MA, USA), and western blotting with anti-AMPK-a1 antibody. The same lysates were verified with anti-AMPK-a1 antibodies (Abcam, Cambridge, MA, USA). anti-TAK1 antibody. In vitro kinase assay for TAK1 and AMPK-a1. Wt THP-1, AMPK- Generation of AMPK-a1-knockdown or TAK1-knockdown THP-1 KD KD cells. Lentiviral particles containing shRNA-targeted human AMPK-a1 (sc-29673-V) a1 , and TAK1 THP-1 cells were treated with or without LPS (100 ng/ml) for or human TAK1 (sc-36606-V) were purchased from Santa Cruz Biotechnology Inc. different times. The kinase assay for TAK1 and AMPK-a1 was performed by (Santa Cruz, CA, USA) 2 Â 10 À 7 cells were cultured in a 24-well plate and infected c-TAK1 Kinase Assay kit (U-TRF#17, PerkinElmer, Branchburg, NJ, USA) and with each rentiviral particle according to the manufacturer’s protocol. Cells were AMPK-a1 Kinase assay kit (U-TRF#12, PerkinElmer) in accordance with the cultured in puromycin (4 mg/ml)-containing medium for 2 weeks to select stable clones. manufacturer’s protocol.

Luciferase reporter assay. The THP-1 cells (2 Â 10 À 5) grown on 12-well Measurement of caspase-3, caspase-8, and caspase-9 activities. plates were transiently transfected using Neon transfection system (Invitrogen, Wt THP-1 and AMPK-a1KD THP-1 cells were cultured in RPMI medium containing Carlsbad, CA, USA), according to the respective manufacturer’s instructions, with 2% FBS. Cells were treated with or without TNF-a (100 ng/ml) for different times, either NF-kB-dependent reporter construct pBIIx-luc or AP-1-dependent reporter harvested, and washed twice with PBS. Caspase-3, caspase-8, and caspase-9 construct AP-1-luc with Renilla luciferase vector (Promega Corporation, Madison, activities were measured using the CaspACE kit (Promega) according to the WI, USA).33 Total DNA concentration in each experiment was maintained by manufacture’s instructions.33

Cell Death and Disease AMPK-a1 as an activating kinase of TAK1 SY Kim et al 12

DNA fragmentation analysis. Cells were homogenized in 1 ml of lysis MAGED1 (PDH 01965A), TNFRFS21 (PDH 00262A), SGK1 (PDH 00387E), buffer (20 mM Tris-HCl, pH 8.0, 5 mM EDTA, 0.5% SDS, 0.5 mg/ml proteinase K) PI3KCB (PDH 00789A), BCL2L2 (PDH 00081E), and BCL2 (PDH 00079B). The and incubated for 15 h at 42 1C under constant agitation. Proteins were then qRT-PCR analysis was performed using Roter-GeneQ (Qiagen) according to the precipitated with 6 M NaCl and centrifuged at 2500 Â g at 4 1C for 15 min. manufacturers’ protocol. Supernatants containing genomic DNA were then treated with RNase A at 37 1C for 30 min. The genomic DNA was precipitated for 3 h at À 70 1C with 2.5 Statistical analysis. Data were presented as mean values±S.D. as volumes of 100% ethanol and 0.2 volumes of 3 M sodium acetate. Samples were indicated and analyzed using Student’s two-tailed t-test. A P-value o0.05 was then centrifuged at 20 800 Â g at 4 1C for 30 min. The resulting pellets were considered to be statistically significant. washed with 70% ethanol and resuspended in 40 ml of nuclease-free water. Genomic DNA extracts (10–20 ml) were run on 1.8% agarose gels and visualized under UV illumination. Conflict of Interest The authors declare no conflict of interest. Cell and apoptosis analysis. Wt THP-1 and AMPK-a1KD THP-1 cells were cultured in RPMI medium containing 2% FBS. Cells were treated with or without TNF-a (100 ng/ml) for different times, harvested, and washed twice with Acknowledgements. This work was supported by a grant from the Korea PBS. The cells were stained with FITC Annexin V Apoptosis Detection Kit Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of (BD Biosciences, San Jose, CA, USA) or BD Cycletest Plus-DNA Reagent kit Korea (A111636). We would like to thank the Hyewa Forum members, namely, (BD Biosciences) in accordance with the manufacturer’s protocol. Samples were Dr. Doo Hyun Chung, Dr. Jun Chang, Dr. You-Me Kim, Dr. Eun Sook Hwang, analyzed with FACSCalibur system and then apoptotic cell death was determined Dr. Eui-Cheol Shin, Dr. Seung-Hyo Lee, Dr. Heung kyu Lee, and Dr. Sang-Jun Ha with CellQuest software (Becton Dickinson, San Jose, CA, USA) or Modfit LT 3.0 for their helpful discussions. software (Becton Dickinson).

Microarray analysis. Total RNA was extracted using Trizol (Invitrogen Life 1. Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N et al. Identification of a Technologies, Carlsbad, CA, USA) and purified using RNeasy columns (Qiagen, member of the MAPKKK family as a potential mediator of TGF-b signal . Valencia, CA, USA) according to the manufacturers’ protocol. After processing Science 1995; 270: 2008–2011. 2. Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is -dependent with DNase digestion, and clean-up procedures, RNA samples were quantified, kinase of MKK and IKK. Nature 2001; 412: 346–351. aliquoted, and stored at À 80 1C until use. For quality control, RNA purity and 3. Shim JH, Xiao C, Paschal AE, Bailey ST, Rao P, Hayden MS et al. TAK1 but not TAB1 or integrity were evaluated by denaturing gel electrophoresis, OD 260/280 ratio, and TAB2, plays an essential role in multiple signaling pathways in vivo. Genes Dev 2005; 19: analyzed on Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). 2668–2681. Total RNA was amplified and purified using the Ambion Illumina RNA amplification 4. Kim SY, Shim JH, Chun E, Lee KY. Reciprocal Inhibition between the transforming kit (Ambion, Austin, TX, USA) to yield biotinylated cRNA according to the growth factor-b-activated kinase 1 (TAK1) and apoptosis signal-regulating kinase 1 manufacturer’s instructions. Briefly, 550 ng of total RNA was reverse-transcribed to (ASK1) mitogen-activated protein kinase kinase kinases and its suppression by TAK1- cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro binding protein 2 (TAB2), an adapter protein for TAK1. J Biol Chem 2012; 287: 3381–3391. transcribed, and labeled with biotin-NTP. After purification, the cRNA was 5. Skaug B, Jiang X, Chen ZJ. The role of ubiquitin in NF-kappaB regulatory pathways. quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, Annu Rev Biochem 2009; 78: 769–796. NC, USA). Typically, 750 ng of labeled cRNA samples were hybridized to each 6. Liu S, Chen ZJ. Expanding role of ubiquitination in NF-kB signaling. Cell Res 2011; 21: humanHT-12 expression v.4 bead array for 16–18 h at 58 1C, according to the 6–21. manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). Detection of array 7. Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev 2004; 18: 2195–2224. signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare 8. West AP, Koblansky AA, Ghosh S. Recognition and signaling by toll-like receptors. Bio-Sciences, Little Chalfont, UK) following the bead array manual. Arrays were Annu Rev Cell Dev Biol 2006; 22: 409–437. scanned with an Illumina bead array Reader confocal scanner according to the 9. Viollet B, Horman S, Leclerc J, Lantier L, Foretz M, Billaud M et al. AMPK inhibition in manufacturer’s instructions. For raw data preparation and statistical analysis, the health and disease. Crit Rev Biochem Mol Biol 2010; 45: 276–295. 10. Carling D, Mayer FV, Sanders MJ, Gamblin SJ. AMP-activated protein kinase: nature’s quality of hybridization and overall chip performance were monitored by visual energy sensor. Nat Chem Biol 2011; 7: 512–518. inspection of both internal quality control checks and the raw scanned data. Raw 11. Corton JM, Gillespie JG, Hardie DG. Role of the AMP-activated protein kinase in the data were extracted using the software provided by the manufacturer (Illumina cellular stress response. Curr Biol 1994; 4: 315–324. GenomeStudio v2009.2 (Gene Expression Module v1.5.4)). Array data were 12. Hardie DG, Carling D. The AMP-activated protein kinase–fuel gauge of the mammalian filtered by detection of P-value o0.05 (similar to signal to noise) in at least 50% cell? Eur J Biochem 1997; 246: 259–273. samples (we applied a filtering criterion for data analysis; higher signal value was 13. Witczak CA, Sharoff CG, Goodyear LJ. AMP-activated protein kinase in skeletal muscle: required to obtain a detection P-value o0.05). Selected gene signal value was from structure and localization to its role as a master regulator of cellular metabolism. transformed by logarithm and normalized by quantile method. The comparative Cell Mol Life Sci 2008; 65: 3737–3755. 14. Salt I, Celler JW, Hawley SA, Prescott A, Woods A, Carling D et al. AMP-activated analysis between test sample and control sample was carried out using fold protein kinase: greater AMP dependence, and preferential nuclear localization, change. 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